Novel retinoid X receptor antagonists: Specific inhibition of retinoid synergism in RXR-RAR heterodimer actions

Article abstract of DOI:10.1021/jm0255320

Several 2-(arylamino)pyrimidine-5-carboxylic acids were designed as novel retinoid X receptor (RXR) antagonists. Compound 6a or 6b alone did not exhibit differentiation-inducing activity toward HL-60 cells and did not affect the activity of a retinoic acid receptor (RAR) agonist, Am80, but did inhibit the synergistic activity of an RXR agonist, PA024 (3), in the presence of Am80. The activity of 6 was ascribed to selective antagonism at the RXR site of RXR-RAR heterodimers.

Full text of DOI:10.1021/jm0255320

© Copyright 2002 by the American Chemical Society
Volume 45, Number 16
August 1, 2002
Letters
well as PPAR agonists.4 In contrast to the various RXR-
selective agonists reported so far,5 only a few RXR
antagonists have been reported. Further, LG100754 (4),
the first reported RXR antagonist, is an RXR ho-
modimer-selective antagonist6 and acts as an agonist
of several RXR heterodimers.7 Previously, we reported
that diazepine derivatives HX531 (5a ) and HX603 (5b)
are RXR antagonists, which can inhibit RXR het-
erodimers.8 Although these diazepines also inhibited
activation of RARs induced by RAR agonists at high
dose, they exhibited antidiabetic and antiobesity activi-
ties by regulating the activities of PPAR-γ-RXR het-
erodimers.9 In view of the clinical potential of antidia-
betic and antiobesity agents, we focused on the develop-
ment of more potent and/or selective RXR antagonists.
Here, we describe novel RXR-selective antagonists,
which can inhibit specifically the synergistic activity of
an RXR agonist on RXR-RAR heterodimer actions.
Ch em istr y. Regarding the design of RXR antagonist
candidates, we used the RXR agonist-antagonist struc-
ture-activity relationship of diazepinylbenzoic acids (5)
to develop the structure of the potent RXR agonist 3.
As reported before, the introduction of a suitable sub-
stituent on the aromatic ring (X in Chart 1) or elonga-
tion of the N-methyl group of the RXR agonist HX600
(5c) resulted in RXR antagonistic activity.8 Of two
antagonists 5a and 5b, the latter, having an N-n-propyl
group, is rather RXR-selective, although its potency is
weaker. Considering that the n-propylamino group of
5b should correspond to the substituent ortho to the
amino group of PA compounds (RO- group in Chart 1),
we synthesized several 2-[N-(3-alkoxy-5,6,7,8-tetrahy-
dro-5,5,8,8-tetramethylnaphthalen-2-yl)-N-methylami-
no]pyrimidine-5-carboxylic acids. The synthetic scheme
is shown in Scheme 1.10 5,6,7,8-Tetrahydro-5,5,8,8-
tetramethyl-2-naphthol (7) was nitrated with HNO3 in
CH2Cl2 to give 8 as a major product (71% yield). After
O-methylation and subsequent hydrogenation of the
nitro group, compound 10 was reacted with ethyl
2-chloropyrimidine-5-carboxylate in the presence of K2-
CO3 at 110 °C to give ethyl 2-(arylamino)pyrimidine-5-
carboxylate (11) in 94% yield. After N-methylation and
Novel Retin oid X Recep tor An ta gon ists:
Sp ecific In h ibition of Retin oid Syn er gism
in RXR-RAR Heter od im er Action s
Bitoku Takahashi,† Kiminori Ohta,† Emiko Kawachi,†
Hiroshi Fukasawa,† Yuichi Hashimoto,‡ and
Hiroyuki Kagechika*,†
Graduate School of Pharmaceutical Sciences, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, J apan,
and Institute of Molecular and Cellular Biosciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-0032, J apan
Received April 23, 2002
Abstr a ct: Several 2-(arylamino)pyrimidine-5-carboxylic acids
were designed as novel retinoid X receptor (RXR) antagonists.
Compound 6a or 6b alone did not exhibit differentiation-
inducing activity toward HL-60 cells and did not affect the
activity of a retinoic acid receptor (RAR) agonist, Am80, but
did inhibit the synergistic activity of an RXR agonist, PA024
(3), in the presence of Am80. The activity of 6 was ascribed to
selective antagonism at the RXR site of RXR-RAR het-
erodimers.
In tr od u ction . Retinoid X receptors (RXRs) exist in
three subtypes (R, â, and γ forms),1 and their endog-
enous ligand has been identified as 9-cis-retinoic acid
(1, Chart 1), which also has high affinities for retinoic
acid receptors (RARs).2 While RXRs act as retinoid
receptors, their key role is in heterodimer formation
with various nuclear receptors, including RARs, vitamin
D3 receptors, thyroid hormone receptors, and peroxi-
some proliferator-activated receptors (PPARs). Studies
using RXR-selective agonists, such as LGD1069 (2) and
PA024 (3), have shown that RXR ligands act in different
ways, depending on the heterodimer partner.1 For
example, RXR agonists alone cannot activate RXR-RAR
heterodimers but enhance the potency of RAR agonists,3
while they can activate PPAR-RXR heterodimers as
* To whom correspondence should be addressed. Phone: +81-3-
5841-4732. Fax: +81-3-5841-4768. E-mail: kage@mol.f.u-tokyo.ac.jp.
† Graduate School of Pharmaceutical Sciences.
‡ Institute of Molecular and Cellular Biosciences.
10.1021/jm0255320 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/09/2002

3328 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Ch a r t 1. Structures of RXR Ligands
Letters
Sch em e 1. Synthetic Scheme of PA451 (6a ) and PA452 (6b)a
a
(a) HNO3, CH2Cl2; (b) NaH, DMF; CH3I; (c) H2, Pd-C, EtOH; (d) ethyl 2-chloropyrimidine-5-carboxylate, K2CO3, 4; (e) BBr3, CH2Cl2;
(f) NaH, DMF, n-C5H11I or n-C6H13Br; (g) KOH, EtOH.
F igu r e 1. Effects of (a) PA451 (6a ), (b) PA452 (6b), and (c) HX531 (5a ) on HL-60 cell differentiation induced by Am80 in the
presence or absence of PA024 (3). Concentration of Am80 is 3 × 10-10 M (open and closed circles) and 1 × 10-10 M (2), and that
of PA024 (3) is zero (O), 3 × 10-10 M (b), and 1 × 10-9 M (2).
subsequent O-demethylation, various O-alkyl groups
were introduced into compound 13. Finally, the ester
group was hydrolyzed under basic conditions.
Ch a r t 2
Resu lts a n d Discu ssion . The biological activity of
the synthesized compounds was first evaluated in terms
of the induced differentiation of human promyelocytic
leukemia cells HL-60.8b,10 Among pyrimidinecarboxylic
acids with various N- and O-alkyl groups, PA451 (6a )
and PA452 (6b) exhibited unique activity, as shown in
Figure 1, compared with 5a . These three compounds did
not elicit differentiation-inducing activity alone or in the
presence of an RXR agonist such as 5c or 3 (data not
shown). When 6a or 6b was added together with a
retinoid, Am80 (an RAR agonist, Chart 2), the dif-
ferentiation-inducing activity of Am80 was not affected
(open circles in Figure 1). However, 6a and 6b inhibited
the differentiation induced by the combination of Am80
and 3, an RXR agonist, in a dose-dependent manner
(closed circles and triangles). 6a similarly inhibited

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3329
F igu r e 2. Transactivation assays in Cos-1 cells transiently transfected with mRXR/(DR1)5-pGL-TK (RXRR, -â, and -γ in parts
a, b, and c, respectively) and hRAR/(TREpal)3-TKLUC (RARR, -â, and -γ in parts d, e, and f, respectively). The vertical scale is
receptor transactivation induced by 1 × 10-8 M Am80 (RARR and -â), 1 × 10-8 M retinoic acid (RARγ), and 1 × 10-8 M PA024
(RXRs). The values were normalized to that obtained when the activator (agonist) alone was added, taken as 100. The horizontal
scale is the molar concentration of the added compound. Added compounds were HX531 (5a , b), PA451 (6a , O), and PA452 (6b,
4).
synergistic combinations of other RAR and RXR ago-
nists (such as Am80 plus 5c, data not shown). Signifi-
cantly, the inhibition by 6a and 6b did not reach the
basal level, and the percentage of differentiated cells
in the presence of a high dose of 6a or 6b was more
than that induced by Am80 alone. For example, addition
of 3 × 10-10 M 3 increased the extent of differentiation
induced by 3 × 10-10 M Am80 from 34% to 73% of the
cells. 6a inhibited this differentiation to 44% (still higher
than 34%, p < 0.05), even at 1 × 10-6 M (Figure 1b,
closed circles). In contrast, 5a inhibited the activity of
both Am80 alone and the combination of Am80 and 3
to the basal level in both cases.
RAR and RXR agonists bind to the heterodimers, 6a and
6b antagonize the RXR agonist at the RXR site, and
consequently, the retinoid synergism by the RXR ago-
nist is decreased. If the RAR agonist occupies the RAR
site of the RXR-RAR heterodimer, the binding of 6a or
6b at the RXR site does not affect the RXR-RAR
actions. Recently, retinoid synergism was explained in
terms of further stabilization of the complex of RAR
agonist-activated RXR-RAR heterodimers and coacti-
vators caused by binding of RXR ligands.11 In light of
this hypothesis, the antagonistic potency of these pyri-
midinecarboxylic acids may be ascribed to loss of this
further stabilization between holoRXR sites and coac-
tivators.
In conclusion, we have developed the first RXR-
selective antagonists that do not affect retinoid actions
but selectively inhibit retinoid synergism by RXR ago-
nists in RAR-RXR heterodimer actions. Since RXRs
form various kinds of nuclear receptor heterodimers and
RXR antagonists improved obesity and insulin resis-
tance through modulation of the PPAR-γ-RXR activity,9
these RXR-selective antagonists may be useful tools for
the elucidation of RXR-related gene networks and for
possible clinical application in the fields of diabetes and
obesity. Further investigations on the biological potency
of RXR-selective antagonists, including the regulatory
mechanisms of various RXR-related heterodimers, are
in progress.
The inhibitory mechanism by 6a and 6b is assumed
to be antagonism at the RXR site of RXR-RAR het-
erodimers. Therefore, their effects on the activation of
retinoid receptors, RARs and RXRs, were investigated
by transient transactivation assay (Figure 2).8b,10 None
of the test compounds 6a , 6b, and 5a , for comparison,
when examined alone, activated any retinoid receptor
(data not shown). These three compounds dose-depen-
dently inhibited the transactivation of all subtypes of
RXRs induced by 1 × 10-8 M 3 (Figure 1a-c). Among
the three compounds, 6a is the most potent inhibitor,
especially for RXRR. Remarkable differences were ob-
served in the effects on RAR transactivations. 5a
inhibited the RAR transactivation induced by 1 × 10-8
M Am80 (R and â subtypes) or by 1 × 10-8 M retinoic
acid (γ subtype), while 6a and 6b were not inhibitory
at concentrations below 1 × 10-6 M.
Ack n ow led gm en t. The authors are grateful to Prof.
P. Chambon, INSERM, France, for his generous supply
of RXR-expression vectors and to Prof. M. Yamamoto,
Institute of Medical Science, The University of Tokyo,
The results obtained here indicate that 6a and 6b do
not activate RXR-RAR heterodimers or affect RXR-
RAR activation by an RAR agonist alone. When both

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J M0255320